Antibodies are normally synthesized by lymphoid cells derived from B lymphocytes in bone marrow. Individual lymphocytes cannot be directly cultured to produce a specific antibody. However, Kohler et al, Nature 256, 495 (1975) demonstrated that a process of somatic cell fusion, specifically between a lymphocyte and myeloma cell, could yield hybrid cells which grow in culture and produce a specific antibody. The hybrid is termed a "hybridoma" cell herein and in the art generally. In a typical fusion procedure, spleen lymphocytes from an animal immunized against a chosen antigen are fused with myeloma cells. The resulting hybridomas are then dispersed in a series of separate culture tubes or microtiter plate wells to screen for cultures providing the desired antibody specific to the antigen. Positive cultures are further diluted to obtain colonies arising from a single cell (or clone). The clones are again screened for production of the desired antibody, which is known as a "monoclonal" antibody in the art.
Monoclonal antibodies are highly specific, being directed to a single determinant on a single antigen, unlike the conventional "polyclonal" antibodies containing molecules specific to various determinants of the antigen. Monoclonal antibodies are useful to improve the selectivity and specificity of diagnostic and therapeutic methods using immunological binding. They are also uncontaminated by other immunoglobulins.
While the production of monoclonal antibodies has been known for some time, there remain difficulties in preparing consistently specific antibodies. For any given case, the choice of animal species and myeloma cell line, and the selection technique used for isolating the desired hybridoma cell line are all important to the outcome.
Horseradish peroxidase (H.sub.2 O.sub.2 : donor oxidoreductase, E.C. 1.11.1.7) has been a useful enzyme label in immunological diagnostic and immunohistochemical reactions for many years. Its principle advantages are its high turnover number and its relative lack of substrate specificity, allowing its activity to be expressed by the generation of a variety of colorimetric, electrochemical or chemiluminescent signals. In addition, horseradish peroxidase is relatively stable to surface denaturation, lyophilization and other potentially hazardous conditions of manufacture or storage.
Both polyclonal and monoclonal antibodies specific to horseradish peroxidase have been developed which have various degrees of inhibitory effect on the enzymatic properties of the enzyme (see for example, Marucci, Immunochemistry, 10, pages 278-280, 1973; Conroy et al, Immunochemistry, 13, pages 599-603, 1976; Conroy et al, Molecular Immunology 19(5), pages 659-663, 1982; Conroy et al, Molecular Immunology 20(6), pages 647-653, 1983; Boot et al, J. Immun. Methods, 103, pages 69-77, 1987; and David A. Tipton, Ph.D. Thesis, Memphis State University, December 1988).
EP-A-0 137 657 (published Apr. 17, 1985) describes the production of monoclonal antibodies specific to horseradish peroxidase which do not interfere with the activity of the enzyme.
There remains a need for "inhibitor" antibodies which shut down substantially all (greater than 95%) of the activity of horseradish peroxidase. There is also a need in antibodies specific to horseradish peroxidase which do not substantially affect enzyme activity but, when bound to the enzyme, completely prevent the binding of inhibiting antibodies to the enzyme.